Angiogenesis using hepatocyte growth factor

ABSTRACT

Methods for enhancing angiogenesis in a mammal using hepatocyte growth factor (“HGF”) are provided. In the methods, HGF can be administered to mammals suffering from, for instance, vascular insufficiency or arterial occlusive disease. Articles of manufacture and kits containing HGF are also provided.

RELATED APPLICATIONS

This application is a non-provisional application filed under 37 CFR1.53(b)(1) and 35 USC 111(a), claiming priority under 35 USC 119 (e) toprovisional application No. 60/004,816 filed Oct. 5, 1995, the contentsof which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions which can beemployed for enhancing angiogenesis in mammals.

BACKGROUND OF THE INVENTION

Hepatocyte growth factor (“HGF”) functions as a growth factor forparticular tissues and cell types. HGF was identified initially as amitogen for hepatocytes [Michalopoulos et al., Cancer Res., 44:4414-4419(1984); Russel et al., J. Cell. Physiol., 119:183-192 (1984); Nakamuraet al., Biochem. Biophys. Res. Comm., 122:1450-1459 (1984)]. Nakamura etal., supra, reported the purification of HGF from the serum of partiallyhepatectomized rats. Subsequently, HGF was purified from rat platelets,and its subunit structure was determined [Nakamura et al., Proc. Natl.Acad. Sci. USA, 83:6489-6493 (1986); Nakamura et al., FEBS Letters,224:311-316 (1987)]. The purification of human HGF (“huHGF”) from humanplasma was first described by Gohda et al., J. Clin. Invest., 81:414-419(1988).

Both rat HGF and huHGF have been molecularly cloned, including thecloning and sequencing of a naturally occurring variant lacking 5 aminoacids designated “deltaS HGF” [Miyazawa et al., Biochem. Biophys. Res.Comm., 163:967-973 (1989); Nakamura et al., Nature, 342:440-443 (1989);Seki et al, Biochem. Biophys. Res. Commun., 172:321-327 (1990); Tashiroet al., Proc. Natl. Acad. Sci. USA, 87:3200-3204 (1990); Okajima et al.,Eur. J. Biochem., 193:375-381 (1990)].

The mature form of huHGF, corresponding to the major form purified fromhuman serum, is a disulfide linked heterodimer derived by proteolyticcleavage of the human pro-hormone between amino acids R494 and V495.This cleavage process generates a molecule composed of an α-subunit of440 amino acids (M_(r) 69 kDa) and a β-subunit of 234 amino acids (M_(r)34 kDa). The nucleotide sequence of the huHGF cDNA reveals that both theα- and the β-chains are contained in a single open reading frame codingfor a pre-pro precursor protein. In the predicted primary structure ofmature huHGF, an interchain S-S bridge is formed between Cys 487 of theα-chain and Cys 604 in the β-chain [see Nakamura et al., Nature, supra].The N-terminus of the α-chain is preceded by 54 amino acids, startingwith a methionine group. This segment includes a characteristichydrophobic leader (signal) sequence of 31 residues and the prosequence.The α-chain starts at amino acid (aa) 55, and contains four kringledomains. The kringle 1 domain extends from about aa 128 to about aa 206,the kringle 2 domain is between about aa 211 and about aa 288, thekringle 3 domain is defined as extending from about aa 303 to about aa383, and the kringle 4 domain extends from about aa 391 to about aa 464of the α-chain.

The definition of the various kringle domains is based on their homologywith kringle-like domains of other proteins (such as prothrombin andplasminogen), therefore, the above limits are only approximate. To date,the function of these kringles has not been determined. The β-chain ofhuHGF shows high homology to the catalytic domain of serine proteases(38% homology to the plasminogen serine protease domain). However, twoof the three residues which form the catalytic triad of serine proteasesare not conserved in huHGF. Therefore, despite its serine protease-likedomain, huHGF appears to have no proteolytic activity, and the preciserole of the β-chain remains unknown. HGF contains four putativeglycosylation sites, which are located at positions 294 and 402 of theα-chain and at positions 566 and 653 of the β-chain.

In a portion of cDNA isolated from human leukocytes, in-frame deletionof 15 base pairs was observed. Transient expression of the cDNA sequencein COS-1 cells revealed that the encoded HGF molecule (deltaS HGF)lacking 5 amino acids in the kringle 1 domain was fully functional [Sekiet al., supra].

A naturally occurring huHGF variant has been identified whichcorresponds to an alternative spliced form of the huHGF transcriptcontaining the coding sequences for the N-terminal finger and first twokringle domains of mature huHGF [Chan et al., Science, 254:1382-1385(1991); Miyazawa et al., Eur. J. Biochem., 197:15-22 (1991)]. Thisvariant, designated HGF/NK2, has been proposed to be a competitiveantagonist of mature huHGF.

Comparisons of the amino acid sequence of rat HGF with that of huHGFhave revealed that the two sequences are highly conserved and have thesame characteristic structural features. The length of the four kringledomains in rat HGF is exactly the same as in huHGF. Furthermore, thecysteine residues are located in exactly the same positions, anindication of similar three-dimensional structures [Okajima et al.,supra; Tashiro et al., supra].

HGF and HGF variants are described further in U.S. Pat. Nos. 5,227,158,5,316,921, and 5,328,837.

The HGF receptor has been identified as the product of the c-Metproto-oncogene [Bottaro et al., Science, 251:802-804 (1991); Naldini etal., Oncogene, 6:501-504 (1991); WO 92/13097 published Aug. 6, 1992; WO93/15754 published Aug. 19, 1993]. The receptor is usually referred toas “Ic-Met” or “p190^(MET)” and typically comprises, in its native form,a 190-kDa heterodimeric (a disulfide-linked 50-kDa α-chain and a 145-kDaβ-chain) membrane-spanning tyrosine kinase protein [Park et al., Proc.Natl. Acad. Sci. USA, 84:6379-6383 (1987)]. Several truncated forms ofthe c-Met receptor have also been described [WO 92/20792; Prat et al.,Mol. Cell. Biol., 11:5954-5962 (1991)].

The binding activity of HGF to its receptor is believed to be conveyedby a functional domain located in the N-terminal portion of the HGFmolecule, including the first two kringles [Matsumoto et al., Biochem.Biophys. Res. Commun., 181:691-699 (1991); Hartmann et al., Proc. Natl.Acad. Sci., 89:11574-11578 (1992); Lokker et al., EMBO J., 11:2503-2510(1992); Lokker and Godowski, J. Biol. Chem., 268:17145-17150 (1991)].The c-Met protein becomes phosphorylated on tyrosine residues of the145-kDa β-subunit upon HGF binding.

Various biological activities have been described for HGF and itsreceptor [see, generally, Chan et al., Hepatocyte Growth Factor-ScatterFactor (HGF-SF) and the C-Met Receptor, Goldberg and Rosen, eds.,Birkhauser verlag-Basel (1993), pp. 67-79]. It has been observed thatlevels of HGF increase in the plasma of patients with hepatic failure[Gohda et al., supra] and in the plasma [Lindroos et al., Hepatol.,13:734-750 (1991)] or serum [Asami et al., J. Biochem., 109:8-13 (1991)]of animals with experimentally induced liver damage. The kinetics ofthis response are usually rapid, and precedes the first round of DNAsynthesis during liver regeneration. HGF has also been shown to be amitogen for certain cell types, including melanocytes, renal tubularcells, keratinocytes, certain endothelial cells and cells of epithelialorigin [Matsumoto et al., Biochem. Biophys. Res. Commun., 176:45-51(1991); Igawa et al., Biochem. Biophys Res. Commun., 174:831-838 (1991);Han et al., Biochem., 30:9768-9780 (1991); Rubin et al., Proc. Natl.Acad. Sci. USA, 88:415-419 (1991)]. Both HGF and the c-Met protooncogenehave been postulated to play a role in microglial reactions to CNSinjuries [DiRenzo et al., Oncogene, 8:219-222 (1993)].

HGF can also act as a “scatter factor”, an activity that promotes thedissociation of epithelial and vascular endothelial cells in vitro[Stoker et al., Nature, 327:239-242 (1987); Weidner et al., J. CellBiol., 111:2097-2108 (1990); Naldini et al., EMBO J., 10:2867-2878(1991); Giordano et al., Proc. Natl. Acad. Sci. USA, 90:649-653 (1993)].Moreover, HGF has recently been described as an epithelial morphogen[Montesano et al., Cell, 67:901-908 (1991)]. Therefore, HGF has beenpostulated to be important in tumor invasion [Comoglio, HepatocyteGrowth Factor-Scatter Factor (HGF-SF) and the C-Met Receptor, Goldbergand Rosen, eds., Birkhauser Verlag-Basel (1993), pp. 131-165].

Therapeutic options for patients with vascular disease, particularlyvascular obstructive disease, are sometimes limited. As Takeshita etal., J. Clin. Invest., 93:662-670 (1994), point out, such patients areoften refractory to conservative measures and typically unresponsive todrug therapy. When vascular obstruction is lengthy and/or widespread,nonsurgical revascularization may not be feasible. Id. Surgical therapy,consisting of arterial bypass and/or amputation, may be complicated by avariable morbidity and mortality, and is often dependent for itsefficacy upon short- and long-term patency of the conduit used. Id.Therapeutic angiogenesis thus constitutes an alternative treatmentstrategy for such patients.

SUMMARY OF THE INVENTION

The invention provides methods for enhancing angiogenesis in a mammalcomprising administering to the mammal an effective amount of HGF. TheHGF alone may be administered to the mammal, or alternatively, may beadministered to the mammal in combination with other therapies and/orpharmacologic agents.

The invention also provides articles of manufacture and kits whichcontain HGF.

Although not being bound by any particular theory, it is presentlybelieved that the HGF can be used to stimulate or enhance angiogenicactivity in patients suffering from vascular insufficiency or limbischemia secondary to arterial occlusive disease.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

As used herein, the terms “hepatocyte growth factor” and “HGF” refer toa growth factor typically having a structure with six domains (finger,Kringle 1, Kringle 2, Kringle 3, Kringle 4 and serine protease domains).Fragments of HGF constitute HGF with fewer domains and variants of HGFmay have some of the domains of HGF repeated; both are included if theystill retain their respective ability to bind a HGF receptor. The terms“hepatocyte growth factor” and “HGF” include hepatocyte growth factorfrom humans (“huHGF”) and any non-human mammalian species, and inparticular rat HGF. The terms as used herein include mature, pre,pre-pro, and pro forms, purified from a natural source, chemicallysynthesized or recombinantly produced. Human HGF is encoded by the cDNAsequence published by Miyazawa et al., 1989, supra, or Nakamura et al.,1989, supra. The sequences reported by Miyazawa et al. and Nakamura etal. differ in 14 amino acids. The reason for the differences is notentirely clear; polymorphism or cloning artifacts are among thepossibilities. Both sequences are specifically encompassed by theforegoing terms. It will be understood that natural allelic variationsexist and can occur among individuals, as demonstrated by one or moreamino acid differences in the amino acid sequence of each individual.The HGF of the invention preferably has at least about 80% sequenceidentity, more preferably at least about 90% sequence identity, and evens more preferably, at least about 95% sequence identity with a nativemammalian HGF. The terms “hepatocyte growth factor” and “HGF”specifically include the deltaS huHGF as disclosed by Seki et al.,supra.

The terms “HGF receptor” and “c-Met” when used herein refer to acellular receptor for HGF, which typically includes an extracellulardomain, a transmembrane domain and an intracellular domain, as well asvariants and fragments thereof which retain the ability to bind HGF. Theterms “HGF receptor” and “c-Met” include the polypeptide molecule thatcomprises the full-length, native amino acid sequence encoded by thegene variously known as p190^(MET). The present definition specificallyencompasses soluble forms of HGF receptor, and HGF receptor from naturalsources, synthetically produced in vitro or obtained by geneticmanipulation including methods of recombinant DNA technology. The HGFreceptor variants or fragments preferably share at least about 65%sequence homology, and more preferably at least about 75% sequencehomology with any domain of the human c-Met amino acid sequencepublished in Rodrigues et al., Mol. Cell. Biol., 11:2962-2970 (1991);Park et al., Proc. Natl. Acad. Sci., 84:6379-6383 (1987); or Ponzetto etal., Oncogene, 6:553-559 (1991).

The term “angiogenesis” is used herein in a broad sense and refers tothe production or development of blood vessels.

The terms “treating,” “treatment,” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy.

The term “mammal” as used herein refers to any mammal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

II. COMPOSITIONS AND METHODS OF THE INVENTION

The present invention provides methods for enhancing angiogenesis usinghepatocyte growth factor, referred to hereinafter as “HGF”. The HGFuseful in the practice of the present invention can be prepared in anumber of ways. For instance, the HGF can be prepared using an isolatedor purified form of HGF. Methods of isolating and purifying HGF fromnatural sources are known in the art. Such isolation and purificationmethods can be employed for obtaining HGF from serum or plasma.Alternatively, HGF can be chemically synthesized and prepared usingrecombinant DNA techniques known in the art and described in furtherdetail the Example below.

The HGF may be from human or any non-human species. For instance, amammal may have administered HGF from a different mammalian species(e.g., rats can be treated with human HGF). Preferably, however, themammal is treated with homologous HGF (e.g., humans are treated withhuman HGF) to avoid potential immune reactions to the HGF. The HGF istypically administered to a mammal diagnosed as having some form ofvascular insufficiency or vascular disease. It is of course contemplatedthat the methods of the invention can be employed in combination withother therapeutic techniques such as surgery.

The HGF is preferably administered to the mammal in apharmaceutically-acceptable carrier. Suitable carriers and theirformulations are described in Remington's Pharmaceutical Sciences, 16thed., 1980, Mack Publishing Co., edited by Oslo et al. Typically, anappropriate amount of a pharmaceutically-acceptable salt is used in theformulation to render the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include liquids such as saline,Ringer's solution and dextrose solution. The pH of the solution ispreferably from about 5 to about 8, and more preferably from about 7 toabout 7.5. The formulation may also comprise a lyophilized powder.Further carriers include sustained release preparations such assemipermeable matrices of solid hydrophobic polymers, which matrices arein the form of shaped articles, e.g., films, liposomes ormicroparticles.

It will be apparent to those persons skilled in the art that certaincarriers may be more preferable depending upon, for instance, the routeof administration and concentration of HGF being administered.

The HGF can be administered to the mammal by injection (e.g.intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. Optionally, the HGFmay be administered by direct intraarterial administration upstream froman occluded artery to optimize concentration and activity of HGF in thelocal circulation of an affected limb.

Effective dosages and schedules for administering the HGF may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of HGF that must be administered will vary depending on, forexample, the mammal which will receive the HGF, the route ofadministration, the particular type of HGF used and other drugs beingadministered to the mammal. A typical daily dosage of the HGF used alonemight range from about 1 μg/kg to up to 100 mg/kg of body weight or moreper day, depending on the factors mentioned above.

HGF may also be administered along with other pharmacologic agents usedto treat the conditions associated with vascular disease such asvascular endothelial growth factor (“VEGF”). The HGF may be administeredsequentially or concurrently with the one or more other pharmacologicagents. The amounts of HGF and pharmacologic agent depend, for example,on what type of drugs are used, the specific condition being treated,and the scheduling and routes of administration.

Following administration of HGF to the mammal, the mammal'sphysiological condition can be monitored in various ways well known tothe skilled practitioner.

In another embodiment of the invention, there are provided articles ofmanufacture and kits containing materials useful for enhancingangiogenesis. The article of manufacture comprises a container with alabel. Suitable containers include, for example, bottles, vials, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for enhancing angiogenesis. The active agent in thecomposition is HGF. The label on the container indicates that thecomposition is used for enhancing angiogenesis, and may also indicatedirections for in vivo use, such as those described above.

The kit of the invention comprises the container described above and asecond container comprising a pharmaceutically-acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All reference citations herein areincorporated by reference.

EXAMPLE

Recombinant human HGF (“rhuHGF”) was produced in CHO cells using aprocedure modified from Naka et al., J. Biol. Chem., 267:20114-20119(1992). rhuHGF-transfected cells were grown in a 400 L bioreactor inmedium containing 2% fetal bovine serum for 8 days. Culture supernatantcontaining rhuHGF was concentrated and clarified, then conditioned bythe addition of solid NaCl to 0.3 M. rhuHGF was then purified in asingle step using cation exchange chromatography. Conditioned,concentrated culture supernatant was loaded onto a column of S-SepharoseFast Flow equilibrated in 20 mM Tris, pH 7.5, 0.3 M NaCl. After washingout unbound protein, rhuHGF was eluted in a linear gradient from 20 mMTris, pH 7.5, 0.3 M NaCl to 20 mM Tris, pH 7.5, 1.2 M NaCl.rhuHGF-containing fractions were pooled based on SDS-PAGE analysis. TheS Sepharose Fast Flow pool was concentrated and exchanged into 20 mMTris, pH 7.5, 0.5 M NaCl by gel filtration on Sephadex G25 to a finalconcentration of about 3-5 mg/ml. A rhuHGF stock solution was thenprepared by diluting the rhuHGF in buffer (0.5% bovine serum albumin,0.05% Tween-20, 0.01% Thimersol in PBS).

The effects of rhuHGF on angiogenesis was tested in a rabbit model ofhindlimb ischemia. The rabbit model was designed to simulate ischemiacharacteristics of patients with severe lower extremity arterialocclusive disease. [Takeshita et al., supra]. The effects of vascularendothelial growth factor (“VEGF”) were also tested and compared torhuHGF. The in vivo experiment was conducted essentially as described inTakeshita et al., supra. One femoral artery was resected in each of 24New Zealand rabbits. Ten days later (Day 0 of study), baselinemeasurements of calf blood pressure (BP) index; angiographic score ofcollateral formation; intravascular Doppler-wire analysis of blood flow;and microsphere-based analysis of muscle perfusion at rest and duringstress were performed. The animals exhibited similar baselinemeasurements.

Each group of animals (8 rabbits/group) then received intra-iliac rhuHGF(500 μg), recombinant human VEGF (“rhuVEGF”) (500 μg) [prepared asdescribed in Ferrara et al., Methods Enzym., 198:391-404 (1991)], orvehicle (saline plus 0.1% rabbit serum albumin), followed by the samedose intravenously at Days 2 and 4 of the study. At Day 30, allmeasurements were repeated, and the animals were sacrificed. Totalmuscle weight of each leg was measured and samples were used forcapillary density. The results at Day 30 are shown below in Table 1.TABLE 1 Day 30 Data Vehicle rhuVEGF rhuHGF Angiographic  0.46 ± 0.06 0.62 ± 0.04†  0.78 ± 0.07†§ Score Capillary 158 ± 12 247 ± 18† 282 ±15†§ Density (/mm2) BP index (%) 51.6 ± 4.5 69.8 ± 3.1† 84.5 ± 1.8†§Blood flow 17.9 ± 1.1 20.6 ± 1.3* 23.4 ± 1.2†§ (ml/min) Muscle perfusion73.2 ± 6.8 88.4 ± 6.6* 99.2 ± 4.5†§ (rest, %) Muscle perfusion 36.6 ±8.8 65.7 ± 7.5† 83.3 ± 6.7†§ (stress, %) Muscle weight 73.0 ± 2.6 87.6 ±2.8* 95.9 ± 5.4†§ (%)% = % of normal limb;*= p < .05 vs vehicle;†= p < .001 vs vehicle;§= p < .05 vs VEGF

The data showed that HGF enhanced collateral vessel formation andregional perfusion, and prevented atrophy. At similar doses in thestudy, the HGF exhibited greater efficiency than VEGF.

1-7. (canceled)
 8. An article of manufacture, comprising: a container; alabel on said container; and a composition comprising an active agentcontained within said container; wherein the composition is effectivefor enhancing angiogeneis, the label on said container indicates thatthe composition can be used for enhancing angiogenesis, and the activeagent in said composition comprises HGF.
 9. The article of manufactureof claim 8 further comprising instructions for administering the HGF toa mammal to enhance angiogenesis.
 10. A kit, comprising: a firstcontainer, a label on said container, and a composition comprising anactive agent contained with said container; wherein the composition iseffective for enhancing angiogenesis, the label on said containerindicates that the composition can be used for enhancing angiogenesis,and the active agent in said composition comprises HGF; a secondcontainer comprising a pharmaceutically-acceptable buffer; andinstructions for using the HGF to enhance angiogenesis.
 11. Apharmaceutical composition for enhancing angiogenesis comprisinghepatocyte growth factor (HGF) in a pharmaceutical carrier acceptablefor intravenous, intraarterial or infusion administration.
 12. Thepharmaceutical composition of claim 11, wherein said carrier comprises abuffering agent.
 13. The pharmaceutical composition of claim 11, whereinsaid HGF is recombinant HGF.
 14. The pharmaceutical composition of claim11, further comprising a pharmacologic agent used to treat conditionsassociated with vascular disease.
 15. The pharmaceutical composition ofclaim 14, wherein said pharmacologic agent is vascular endothilialgrowth factor (VEGF).